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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Escherichia coli DNA-directed RNA polymerase is shown to contain a novel phosphorolytic error correction activity which removes erroneous nucleotides, as rNDPs, from the 3'-end of the growing transcript. The activity we describe is biochemically similar to polynucleotide phosphorylase (PNP), yet in contrast to PNP is activated by Mn2+. We demonstrate that the activity, which is mediated by Pi, is dependent on the presence of an incorrectly incorporated nucleotide at the leading 3'-end of the transcript. The correction activity we describe exhibits a 4 x 10(4)-fold preference for the excision of incorrect nucleotides from the transcript. These findings suggest the possibility that RNA phosphorolysis may play a critical role in the process of transcriptional proofreading.
Mol Microbiol 1994 Apr
PMID:Phosphorolytic error correction during transcription. 752 Jan 15

Catalyzed polymerization reactions represent a primary anabolic activity of all cells. It can be assumed that early cells carried out such reactions, in which macromolecular catalysts were encapsulated within some type of boundary membrane. In the experiments described here, we show that a template-independent RNA polymerase (polynucleotide phosphorylase) can be encapsulated in dimyristoyl phosphatidylcholine vesicles without substrate. When the substrate adenosine diphosphate (ADP) was provided externally, long-chain RNA polymers were synthesized within the vesicles. Substrate flux was maximized by maintaining the vesicles at the phase transition temperature of the component lipid. A protease was introduced externally as an additional control. Free enzyme was inactivated under identical conditions. RNA products were visualized in situ by ethidium bromide fluorescence. The products were harvested from the liposomes, radiolabeled, and analyzed by polyacrylamide gel electrophoresis. Encapsulated catalysts represent a model for primitive cellular systems in which an RNA polymerase was entrapped within a protected microenvironment.
J Mol Evol 1994 Dec
PMID:Production of RNA by a polymerase protein encapsulated within phospholipid vesicles. 752 10

RNA-OUT, the 69-nucleotide antisense RNA that regulates Tn10/IS10 transposition folds into a simple stem-loop structure. The unusually high metabolic stability of RNA-OUT is dependent, in part, on the integrity of its stem-domain: mutations that disrupt stem-domain structure (Class II mutations) render RNA-OUT unstable, and restoration of structure restores stability. Indeed, there is a strong correlation between the thermodynamic and metabolic stabilities of RNA-OUT. We show here that stem-domain integrity determines RNA-OUT's resistance to 3' exoribonucleolytic attack: Class II mutations are almost completely suppressed in Escherichia coli cells lacking its principal 3' exoribonucleases, ribonuclease II (RNase II) and polynucleotide phosphorylase (PNPase). RNase II and PNPase are individually able to degrade various RNA-OUT species, albeit with different efficiencies: RNA-OUT secondary structure provides greater resistance to RNase II than to PNPase. Surprisingly, RNA-OUT is threefold more stable in wild-type cells than in cells deficient for RNase II activity, suggesting that RNase II somehow lessens PNPase attack on RNA-OUT. We discuss how this might occur. We also show that wild-type RNA-OUT stability changes only two-fold across the normal range of physiological growth temperatures (30-44 degrees C) in wild-type cells, which has important implications for IS10 biology.
Mol Microbiol 1994 Sep
PMID:Decay of the IS10 antisense RNA by 3' exoribonucleases: evidence that RNase II stabilizes RNA-OUT against PNPase attack. 753 7

Stem-loop structures can protect upstream mRNA from degradation by impeding the processive activities of 3'-5' exoribonucleases. The ability of such structures to impede exonuclease activity in vitro is insufficient to account for the stability they can confer on mRNA in vivo. In this study we identify a factor from Escherichia coli which specifically impedes the processive activity of the 3'-5' exonuclease PNPase at stem-loop structures in vitro. This factor can, potentially, reconcile the apparent discrepancy between the ability of 3' stem-loop structures to stabilize upstream mRNA in vitro and in vivo. Its mechanism of action, and possible role in regulating mRNA degradation, is discussed.
Mol Microbiol 1994 Nov
PMID:mRNA degradation in Escherichia coli: a novel factor which impedes the exoribonucleolytic activity of PNPase at stem-loop structures. 753 70

mRNA degradation in Escherichia coli is mediated by a combination of exo- and endoribonucleases. We present evidence for a multiprotein complex which includes at least two enzymes that play important roles in mRNA degradation: the exoribonuclease polynucleotide phosphorylase (PNPase) and the endoribonuclease RNase E. An activity which impedes the processive activity of PNPase at stem-loop structures also appears to be associated with the complex. This complex is estimated to have a molecular mass of about 500 kDa and includes several additional polypeptides whose functions are unknown. The identification of a complex which includes several activities associated with mRNA degradation has implications for the mechanisms and co-ordinated control of mRNA degradation.
Mol Microbiol 1994 Nov
PMID:A protein complex mediating mRNA degradation in Escherichia coli. 789 59

In the presence of Mg2+ ions, polynucleotide phosphorylase (PNPase, EC 2.7.7.8) is known to synthesize RNA-like polymers using ribonucleoside-5'-diphosphate (NDP) substrates but to be unable to utilize deoxyribonucleoside substrates. Our experiments show that when MgCl2 is replaced by FeCl3, PNPase becomes able to synthesize deoxyheteropolymers using deoxyribonucleoside-5'-diphosphates (dNDPs). The deoxyheteropolymer formed from the four dNDPs is degraded by pancreatic DNase, but not by RNase, and is readily used as a template by DNA-dependent DNA polymerase. Synthesis of this DNA-like polymer is accomplished de novo without the help of any primer or preexisting template. What is more, dA/dG and dC/dT ratios of polymers synthesized by different bacterial PNPases closely match ratios found in DNA of the bacterial species the enzyme came from.
J Mol Evol 1996 May
PMID:De Novo Synthesis of DNA-Like Molecules by Polynucleotide Phosphorylase In Vitro 866 1

PNPase and RNase II are the key regulatory exonucleases controlling mRNA decay in Escherichia coli. The rnb transcripts were found to proceed through the terminator and PNPase was found to be involved in the 3' to 5' degradation of rnb mRNA. Analysis of these longer 3' termini revealed that they are located in UA-rich regions. Comparison of single and double mutants suggested that PNPase and RNase II could have different roles in the degradation of these unstructured regions. We have shown that RNase II levels can vary over a fivefold range in haploid cells and that its expression depends on PNPase levels. PNPase-deficient strains were found to have a 2-2.5-fold increase in RNase II activity, while PNPase-overproducing strains reduced the rnb message and RNase II levels. Conversely, the amount of PNPase in the rnb deletion strain was approximately twofold higher than that in the wild-type strain. These observations suggest that the two main exonucleases are inter-regulated through a fine tuning mechanism. We discuss the implications of these results with regard to mRNA degradation and cell metabolism.
Mol Microbiol 1996 Jun
PMID:PNPase modulates RNase II expression in Escherichia coli: implications for mRNA decay and cell metabolism. 880 56

A phosphate-dependent exonuclease activity was identified in purified protein fractions from Bacillus subtilis that were selected for binding to poly(I)-poly(C) agarose. Based on the characteristics of the degradation products and the absence of this activity in a pnpA strain, which contains a transposon insertion in the B. subtilis PNPase gene (Luttinger et al., 1996--accompanying paper), this exonuclease activity was shown to be due to polynucleotide phosphorylase (PNPase). Processive 3'-to-5' exonucleolytic degradation of an SP82 phage RNA substrate was stalled at a particular site. Structure probing of the RNA showed that the stall site was downstream of a particular stem-loop structure. A similar stall site was observed for an RNA that comprised the intergenic region between the B. subtilis rpsO and pnpA genes. The ability to initiate degradation of a substrate that had a stem structure at its 3' end differed for the B. subtilis and Escherichia coli PNPase enzymes.
Mol Microbiol 1996 Jan
PMID:In vitro processing activity of Bacillus subtilis polynucleotide phosphorylase. 882 78

comR (pnpA) is a newly identified gene in Bacillus subtilis that is necessary for the expression of late competence genes. Transformability of a comR (pnpA) mutant is 1-5% of that seen in comR+ strains. Cloning and sequencing identified ComR as polynucleotide phosphorylase (PNPase). The PNPase amino acid sequence has 50% identity and 67% similarity with the Escherichia coli enzyme. Enzymatic assays show that this is the only PNPase activity in B. subtilis. comR (pnpA) is necessary for comG-lacZ and comK-lacZ expression, but this requirement is bypassed by a mecA disruption. In B. subtilis, the loss of PNPase has little effect on expression from a fusion of the srfA promoter directly to lacZ, but is necessary for normal expression from certain srfA-lacZ fusions that include portions of the normal srfA transcript. When a srfA-lacZ translational fusion is tested in isogenic pnpA+ and pnpA derivatives of E. coli, lower expression is seen in the pnpA mutant. Since expression from lacZ fusions to comA, sinR, and mecA appeared similar in the B. subtilis pnpA and pnpA+ strains, the loss of PNPase does not have a strong general effect on gene expression. These results suggest that PNPase may be necessary for modification of the srfA transcript in order to activate translation or stabilize the transcript, and that this may be necessary for competence development. This is the first evidence of post-transcriptional effects on the development of competence in B. subtilis.
Mol Microbiol 1996 Jan
PMID:Polynucleotide phosphorylase is necessary for competence development in Bacillus subtilis. 882 79

The monocistronic transcript of rpsO undergoes an endonucleolytic cleavage downstream of the coding sequence, which removes the hairpin of the transcription terminator and initiates the rapid degradation of the message. We demonstrate here that the two rne-dependent cleavages, on both sides of the transcription terminator, are catalysed by RNase E in vitro and that the RNase E-processed rpsO message is rapidly degraded by polynucleotide phosphorylase, while RNase II produces stable decay intermediates. Moreover, we show that RNase E cuts in vitro the coding sequence of the rpsO mRNA at several sites which are not detected in vivo.
Mol Microbiol 1996 Mar
PMID:Polynucleotide phosphorylase is required for the rapid degradation of the RNase E-processed rpsO mRNA of Escherichia coli devoid of its 3' hairpin. 883 Feb 80


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